To understand the regulation and mechanism of smooth muscle contraction and how actin and myosin interact in nonmuscle cells, we have been using various assays of myosin function. One of these, the in vitro motility assay, involves the visualization, in the fluorescent microscope, of the ATP-dependent movement of fluorescently-labeled actin filaments over a surface coated with myosin molecules. The movement of both smooth and nonmuscle myosin is dependent on phosphorylation of the regulatory light chain. Unphosphorylated myosin does not support movement, but does interact weakly with actin. Smooth and nonmuscle myosins translocate actin filaments slowly compared to skeletal muscle myosin, reflecting their quantitively different kinetics. If the slowly cycling smooth or nonmuscle myosin is mixed with the faster cycling skeletal muscle myosin, the composite velocity is dominated by the slower cycling myosin. Interestingly, the noncycling unphosphorylated smooth and nonmuscle myosin can also slow the faster cycling myosins, presumably by creating an internal load through its weak interactions with actin.